Potent compstatin analogs

ABSTRACT

Compounds comprising peptides and peptidomimetics capable of binding C3 protein and inhibiting complement activation are disclosed. These compounds display greatly improved complement activation-inhibitory activity as compared with currently available compounds.

Continuation of U.S. application Ser. No. 11/605,182, filed Nov. 28,2006 and issued Feb. 15, 2011 as U.S. Pat. No. 7,888,323, which claimsbenefit of U.S. Provisional Application No. 60/740,205, filed Nov. 28,2005, the entire contents of each of which are incorporated by referenceherein.

GOVERNMENT SUPPORT

Pursuant to 35 U.S.C. §202(c), it is acknowledged that the United Statesgovernment may have certain rights in the invention described herein,which was made in part with funds from the National Institutes of Healthunder Grant No. GM 62134.

FIELD OF THE INVENTION

This invention relates to activation of the complement cascade in thebody. In particular, this invention provides peptides andpeptidomimetics capable of binding the C3 protein and inhibitingcomplement activation.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety. Full citations for publications notcited fully within the specification are set forth at the end of thespecification.

The complement system is the first line of immunological defense againstforeign pathogens. Its activation through the classical, alternative orlectin pathways leads to the generation of anaphylatoxic peptides C3aand C5a and formation of the C5b-9 membrane attack complex. Complementcomponent C3 plays a central role in activation of all three pathways.Activation of C3 by complement pathway C3 convertases and its subsequentattachment to target surface leads to assembly of the membrane attackcomplex and ultimately to damage or lysis of the target cells. C3 isunique in that it possesses a rich architecture that provides amultiplicity of diverse ligand binding sites that are important inimmune surveillance and immune response pathways.

Inappropriate activation of complement may lead to host cell damage.Complement is implicated in several disease states, including variousautoimmune diseases, and has been found to contribute to other clinicalconditions such as adult respiratory syndrome, heart attack, rejectionfollowing xenotransplantation and burn injuries. Complement-mediatedtissue injury has also been found to result from bioincompatibilitysituations such as those encountered in patients undergoing dialysis orcardiopulmonary bypass.

Complement-mediated tissue injuries are directly mediated by themembrane attack complex, and indirectly by the generation of C3a andC5a. These peptides induce damage through their effects on variouscells, including neutrophils and mast cells. In vivo, regulation ofcomplement at the C3 and C5 activation steps is provided by both plasmaand membrane proteins. The plasma protein inhibitors are factor H andC4-binding protein, and the regulatory membrane proteins located on cellsurfaces are complement receptors 1 (CR1), decay-accelerating factor(DAF), and membrane cofactor protein (MCP). These proteins inhibit theC3 and C5 convertases (multi-subunit proteases), by promotingdissociation of the multisubunit complexes and/or by inactivating thecomplexes through proteolysis (catalyzed by factor I). Severalpharmacological agents that regulate or modulate complement activityhave been identified by in vitro assay, but most have been shown in vivoto be of low activity or toxic.

To date, there are no inhibitors of complement activation approved foruse in the clinic, though certain candidates for clinical use exist,specifically, a recombinant form of complement receptor 1 known assoluble complement receptor 1 (sCR1) and a humanized monoclonal anti-05antibody (5G1.1-scFv). Both of these substances have been shown tosuppress complement activation in in vivo animal models (Kalli K R etal., 1994; and, Wang et al., 1996). However, each substance possessesthe disadvantage of being a large molecular weight protein (240 kDa and26 kDa, respectively) that is difficult to manufacture and must beadministered by infusion. Accordingly, recent research has emphasizedthe development of smaller active agents that are easier to deliver,more stable and less costly to manufacture.

U.S. Pat. No. 6,319,897 to Lambris et al. describes the use of aphage-displayed combinatorial random peptide library to identify a27-residue peptide that binds to C3 and inhibits complement activation.This peptide was truncated to a 13-residue cyclic segment thatmaintained complete activity, which is referred to in the art ascompstatin. Compstatin inhibits the cleavage of C3 to C3a and C3b by C3convertases. Compstatin has been tested in a series of in vitro, invivo, ex vivo, and in vivo/ex vivo interface experiments, and has beendemonstrated to: (1) inhibit complement activation in human serum (SahuA et al., 1996); (2) inhibit heparin/protamine-induced complementactivation in primates without significant side effects (Soulika A M etal., 2000); (3) prolong the lifetime of a porcine-to-human xenograftperfused with human blood (Fiane A E et al., 1999a; Fiane A E et al.,1999b; and, Fiane A E et al., 2000); (4) inhibit complement activationin models of cardio-pulmonary bypass, plasmapheresis, and dialysisextra-corporeal circuits (Nilsson B et al., 1998); and (5) possess lowtoxicity (Furlong S T et al., 2000).

Compstatin is a peptide comprising the sequence ICVVQDWGHHRCT-NH₂ (SEQID NO:1), where Cys2 and Cys12 form a disulfide bridge. Itsthree-dimensional structure was determined using homonuclear 2D NMRspectroscopy in combination with two separate experimentally restrainedcomputational methodologies. The first methodology involved distancegeometry, molecular dynamics, and simulated annealing (Morikis D et al.,1998; WO99/13899) and the second methodology involved globaloptimization (Klepeis et al., J. Computational Chem., 20:1344-1370,1999). The structure of compstatin revealed a molecular surface thatcomprises of a polar patch and a non-polar patch. The polar partincludes a Type I β-turn and the non-polar patch includes the disulfidebridge. In addition, a series of analogs with alanine replacements (analanine scan) was synthesized and tested for activity, revealing thatthe four residues of the β-turn and the disulfide bridge with thesurrounding hydrophobic cluster play important roles in compstatin'sinhibitory activity (Morikis et al., 1998; WO99/13899).

Using a complement activity assay comprising measuring alternativepathway-mediated erythrocyte lysis, the IC₅₀ of compstatin has beenmeasured as 12 μM. Certain of the analogs previously tested havedemonstrated activity equivalent to or greater than that of compstatin.Published International application No. WO2004/026328 disclosescompstatin analogs and mimetics with variations at the N- and C-termini,and at positions 4 and 9, which imparted improved activity in theaforementioned assay. Improvements of up to 99-fold over compstatin werereported for certain analogs (see also, Mallik et al., 2005). Thedevelopment of compstatin analogs or mimetics with even greater activitywould constitute a significant advance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs and mimetics of thecomplement-inhibiting peptide, compstatin (HOOC-ICVVQDWGHHRCT-NH₂; SEQID NO:1), which have improved complement-inhibiting activity as comparedto compstatin.

In one aspect, the invention features a compound that inhibitscomplement activation, which comprises a peptide having a sequence:

(SEQ ID NO: 26) Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5;wherein:

Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprisingGly-Ile;

Xaa2 is Trp or an analog of Trp, wherein the analog of Trp has increasedhydrophobic character as compared with Trp, with the proviso that, ifXaa3 is Trp, Xaa2 is the analog of Trp;

Xaa3 is Trp or an analog of Trp comprising a chemical modification toits indole ring wherein the chemical modification increases the hydrogenbond potential of the indole ring;

Xaa4 is His, Ala, Phe or Tip;

Xaa5 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide comprising Thr-Asn, ora dipeptide comprising Thr-Ala, or a tripeptide comprising Thr-Ala-Asn,wherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Glyor Asn optionally is replaced by —NH₂; and

the two Cys residues are joined by a disulfide bond.

In certain embodiments, Xaa2 participates in a nonpolar interaction withC3. In other embodiments, Xaa3 participates in a hydrogen bond with C3.In other embodiments, Xaa2 participates in a nonpolar interaction withC3, and Xaa3 participates in a hydrogen bond with C3.

In various embodiments, the analog of Trp of Xaa2 is a halogenatedtrpytophan, such as 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan. Inother embodiments, the Trp analog at Xaa2 comprises a lower alkoxy orlower alkyl substituent at the 5 position, e.g., 5-methoxytryptophan or5-methyltryptophan. In other embodiments, the Trp analog at Xaa2comprises a lower alkyl or a lower alkenoyl substituent at the 1position, with exemplary embodiments comprising 1-methyltryptophan or1-formyltryptophan. In other embodiments, the analog of Trp of Xaa3 is ahalogenated tryptophan such as 5-fluoro-1-tryptophan or6-fluoro-1-tryptophan.

In certain embodiments, Xaa2 comprises a lower alkenoyl or lower alkylsubstituent at the 1 position of tryptophan, Xaa3 optionally comprises ahalogenated tryptophan and Xaa4 comprises Alanine. In particularembodiments, Xaa2 is 1-methyltryptophan or 1-formyltryptophan and Xaa3optionally comprises 5-fluoro-1-tryptophan. Some exemplary compounds ofthe invention comprise any of SEQ ID NOS: 15-25.

In some embodiments, the compound comprises a peptide produced byexpression of a polynucleotide encoding the peptide. In otherembodiments, the compound is produced at least in part by peptidesynthesis. A combination of synthetic methods can also be used.

In certain embodiments, the compstatin analogs are, wherein the compoundis PEGylated, as exemplified by the compound comprising SEQ ID NO:36.

In other embodiments, the compstatin analog further comprises anadditional peptide component that extends the in vivo retention of thecompound. For example, the additional peptide component can be analbumin binding peptide. One exemplary compstatin-albumin bindingpeptide conjugate comprises SEQ ID NO:39.

Another aspect of the invention features a compound that inhibitscomplement activation, comprising a non-peptide or partial peptidemimetic of SEQ ID NO:26 or any of the other sequences of analogs andconjugates described hereinabove. These non-peptide or partial peptidemimetics are designed to bind C3 and inhibit complement activation withat least 100-fold greater activity than does a peptide comprising SEQ IDNO:1 under equivalent assay conditions.

The compstatin analogs, conjugates and mimetics of the invention are ofpractical utility for any purpose for which compstatin itself isutilized, as known in the art and described in greater detail herein.Certain of these uses involve the formulation of the compounds intopharmaceutical compositions for administration to a patient. Suchformulations may comprise pharmaceutically acceptable salts of thecompounds, as well as one or more pharmaceutically acceptable diluents,carriers excipients, and the like, as would be within the purview of theskilled artisan.

Various features and advantages of the present invention will beunderstood by reference to the detailed description, drawings andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Activity of expressed compstatin and its analogs. Plots ofpercent complement inhibition versus peptide concentration forAc-V4W/H9A (SEQ ID NO:5) (squares) and expressed compstatin withtryptophan (SEQ ID NO:15) (circles), 5-fluoro-tryptophan (SEQ ID NO:16)(triangles), 6-fluoro-tryptophan (SEQ ID NO:17 (stars),5-hydroxy-tryptophan (SEQ ID NO:27) (hexagons), 7-aza-tryptophan (SEQ IDNO: 28) (diamonds).

FIG. 2. Activity of synthetic compstatin analogs. Plots of percentcomplement inhibition versus peptide concentration for Ac-V4W/H9A (SEQID NO:5) (squares) and the compstatin analogs with 5-fluoro-l-tryptophanincorporation at position 4 (SEQ ID NO:18) (circles), position 7 (SEQ IDNO:19) (triangles), both positions 4 and 7 (SEQ ID NO:20) (diamonds).

FIG. 3. Activity of additional synthetic compstatin analogs. Plots ofpercent complement inhibition vs. peptide concentration for (A)Ac-V4W/H9A (SEQ ID NO:5) (triangles) compared to Ac-V4(5f-l-W)/H9A (SEQID NO:18) (inverted triangle), Ac-V4(5-methyl-W)/H9A (SEQ ID NO:22)(circles), Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (diamonds),Ac-V4(2-Nal)/H9A (SEQ ID NO:7) (squares); (B) Ac-V4W/H9A (SEQ ID NO:5)(triangles) compared to Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) (hexagons);and, (C) wild-type compstatin (SEQ ID NO:1) (triangles) compared toAc-V4(1-methyl-W)/W7(5f-l-W)/H9A (SEQ ID NO:24) (triangles pointingleft).

FIG. 4. Thermodynamic characterization of the interaction of additionalcompstatin analogs with C3. ITC data representing the binding of (A)Ac-V4W/H9A (SEQ ID NO:5); (B) Ac-V4(5f-l-W)/H9A (SEQ ID NO:18); (C)Ac-V4(5-methyl-W)/H9A (SEQ ID NO:22); (D) Ac-V4(1-methyl-W)/H9A (SEQ IDNO:23); (E) Ac-V4(2-Nal)/H9A (SEQ ID NO:7); and, (F)Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) to C3. The plots were obtained byfitting the corrected raw data to “one set of sites” model in Origin 7.0

FIG. 5. Plots showing the relation between hydrophobicity of the analogsdenoted by log P and the inhibitory constant (A), entropy denoted by−TΔS (B) and the binding constant (C).

FIG. 6. Activity of an additional synthetic compstatin analog. Plots ofpercent complement inhibition vs. peptide concentration forAc-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles) and Ac-V4(1-formyl-W)/H9A(SEQ ID NO:25) (squares)

FIG. 7. Activity of the PEGylated compstatin analog. Plots of percentcomplement inhibition vs. peptide concentration forAc-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles) andAc-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36) (squares).

FIG. 8. Activity of the albumin binding protein-conjugated compstatinanalog. Plots of percent complement inhibition vs. peptide concentrationfor Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles) and the fusionpeptide (Ac-ICV(1MeW)QDWGAHRCTRLIEDICLPRWGCLWEDD-NH₂) (SEQ ID NO:39)(squares).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

DEFINITIONS

The following abbreviations may be used in the specification andexamples: Ac, acetyl group; NH₂, amide; MALDI, matrix-assisted laserdesorption ionization; TOF, time of flight; ITC, isothermal titrationcalorimetry; HPLC, high performance liquid chromatography; NA, notactive; dT, D-threonine; 2-Nal, 2-napthylalanine; 1-Nal,1-napthylalanine; 2-Igl, 2-indanylglycine; Dht, dihydrotryptophan; Bpa,4-benzoyl-L-phenylalanine; 5f-l-W, 5-fluoro-l-tryptophan; 6f-l-W,6-fluoro-l-tryptophan; 5-OH-W, 5-hydroxytryptophan; 5-methoxy-W,5-methoxytryptophan; 5-methyl-W, 5-methyltryptophan; 1-methyl-W,1-methyltryptophan; amino acid abbreviations use the standard three- orsingle-letter nomenclature, for example Trp or W for tryptophan.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, in some embodiments ±5%, in someembodiments ±1%, and in some embodiments ±0.1% from the specified value,as such variations are appropriate to make and used the disclosedcompounds and compositions.

The terms “pharmaceutically active” and “biologically active” refer tothe ability of the compounds of the invention to bind C3 or fragmentsthereof and inhibit complement activation. This biological activity maybe measured by one or more of several art-recognized assays, asdescribed in greater detail herein.

As used herein, “alkyl” refers to an optionally substituted saturatedstraight, branched, or cyclic hydrocarbon having from about 1 to about10 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 1 to about 7carbon atoms being preferred. Alkyl groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl,cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl,and 2,3-dimethylbutyl. The term “lower alkyl” refers to an optionallysubstituted saturated straight, branched, or cyclic hydrocarbon havingfrom about 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein).Lower alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl and neopentyl.

As used herein, “halo” refers to F, Cl, Br or I.

As used herein, “alkanoyl”, which may be used interchangeably with“acyl”, refers to an optionally substituted a straight or branchedaliphatic acylic residue having from about 1 to about 10 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 7 carbon atomsbeing preferred. Alkanoyl groups include, but are not limited to,formyl, acetyl, propionyl, butyryl, isobutyryl pentanoyl, isopentanoyl,2-methyl-butyryl, 2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl,and the like. The term “lower alkanoyl” refers to an optionallysubstituted straight or branched aliphatic acylic residue having fromabout 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein.Lower alkanoyl groups include, but are not limited to, formyl, acetyl,n-propionyl, iso-propionyl, butyryl, iso-butyryl, pentanoyl,iso-pentanoyl, and the like.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl radicals bearing an arylsubstituent and have from about 6 to about 20 carbon atoms (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred. Aralkyl groups can be optionally substituted. Non-limitingexamples include, for example, benzyl, naphthylmethyl, diphenylmethyl,triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy, among others.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen at selected locations on a molecule.Exemplary substituents include, for example, halo, alkyl, cycloalkyl,aralkyl, aryl, sulfhydryl, hydroxyl (—OH), alkoxyl, cyano (—CN),carboxyl (—COOH), acyl (alkanoyl: —C(═O)R); aminocarbonyl (—C(═O)NH₂),—N-substituted aminocarbonyl (—C(═O)NHR″), CF₃, CF₂CF₃, and the like. Inrelation to the aforementioned substituents, each moiety R″ can be,independently, any of H, alkyl, cycloalkyl, aryl, or aralkyl, forexample.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

“Hydrophobic” or “nonpolar” are used synonymously herein, and refer toany inter- or intra-molecular interaction not characterized by a dipole.

As used herein, “pi character” refers to the capacity of compstatin toparticipate in a pi bond with C3. Pi bonds result from the sidewaysoverlap of two parallel p orbitals.

As used herein, “hydrogen bond potential” refers to the capacity ofcompstatin to participate in an electrostatic attraction with C3involving electronegative moieties on the modified tryptophan residuesor tryptophan analogs on compstatin and hydrogen atoms on C3. Anon-limiting example of such an electronegative moiety is a fluorineatom.

“PEGylation” refers to the reaction in which at least one polyethyleneglycol (PEG) moiety, regardless of size, is chemically attached to aprotein or peptide to form a PEG-peptide conjugate. “PEGylated meansthat at least one PEG moiety, regardless of size, is chemically attachedto a peptide or protein. The term PEG is generally accompanied by anumeric suffix that indicates the approximate average molecular weightof the PEG polymers; for example, PEG-8,000 refers to polyethyleneglycol having an average molecular weight of about 8,000.

As used herein, “pharmaceutically-acceptable salts” refers toderivatives of the disclosed compounds wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically-acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Thus, the term “acid addition salt” refers to the correspondingsalt derivative of a parent compound that has been prepared by theaddition of an acid. The pharmaceutically-acceptable salts include theconventional salts or the quaternary ammonium salts of the parentcompound formed, for example, from inorganic or organic acids. Forexample, such conventional salts include, but are not limited to, thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Certain acidic orbasic compounds of the present invention may exist as zwitterions. Allforms of the compounds, including free acid, free base, and zwitterions,are contemplated to be within the scope of the present invention.

DESCRIPTION

In accordance with the present invention, information about thebiological and physico-chemical characteristics of compstatin have beenemployed to design compstatin analogs with significantly improvedactivity compared to the parent compstatin peptide. In some embodiments,the analogs have at least 50-fold greater activity than does compstatin.In other embodiments, the analogs have 60-, 65-, 70-, 75-, 80-, 85-,90-, 95-, 100-, 105-, 110-, 115-, 120-, 125-, or 130-fold or greateractivity than does compstatin. In still other embodiments, the analogshave, 135-, 140-, 145-, 150-, 155-, 160-, 165-, 170-, 175-, 180-, 185-,190-, 195-, 200-, 205-, 210-, 215-, 220-, 225-, 230-, 235-, 240-, 245-,250-, 255-, 260-, 265-fold or greater activity than does compstatin, ascompared utilizing the assays described in the examples.

Compstatin analogs synthesized in accordance with other approaches havebeen shown to possess somewhat improved activity as compared with theparent peptide, i.e., up to about 99-fold (Mallik, B. et al, 2005,supra; WO2004/026328). The analogs produced in accordance with thepresent invention possess even greater activity than either the parentpeptide or analogs thereof produced to date, as demonstrated by in vitroassays as shown in the figures and in the Examples herein.

Table 1B shows amino acid sequence and complement inhibitory activitiesof compstatin and selected analogs with significantly improved activity.The selected analogs are referred to by specific modifications ofdesignated positions (1-13) as compared to the parent peptide,compstatin (SEQ ID NO:1) and to the peptides of SEQ NOS: 2-14, shown inTable 1A, which were described in WO2004/026328. The peptides of SEQ IDNOS: 15-24 are representative of modifications made in accordance withthe present invention, resulting in significantly more potent compstatinanalogs. As described in greater detail below, it will be understoodthat certain of the modifications made to tryptophan at position 4 asset forth in SEQ ID NOS: 2-13 may be combined with a tryptophan analogsubstitution at position 7, to form yet additional potent compstatinanalogs.

TABLE 1 SEQ Activity ID over Peptide Sequence NO: compstatinA. Compstatin and Previously Described Analogs CompstatinH-ICVVQDWGHHRCT-CONH2 1 * Ac-compstatin Ac-ICVVQDWGHHRCT-CONH2 2   3 ×more Ac-V4Y/H9A Ac-ICV Y QDWG A HRCT-CONH2 3  19 × more Ac-V4W/H9A −OHAc-ICV W QDWG A HRCT-COOH 4  25 × more Ac-V4W/H9A Ac-ICV W QDWG AHRCT-CONH2 5  55 × more Ac-V4W/H9A/T13dT −OH Ac-ICV W QDWG A HRC dT-COOH 6  55 × more Ac-V4(2-Nal)/H9A Ac-ICV( 2-Nal )QDWG A HRCT-CONH2 7 99 × more Ac V4(2-Nal)/H9A −OH Ac-ICV( 2-Nal )QDWG A HRCT-COOH 8  39 ×more Ac V4(1-Nal)/H9A −OH Ac-ICV( 1-Nal )QDWG A HRCT-COOH 9  30 × moreAc-V4Igl/H9A Ac-ICV(2- Igl )QDWG A HRCT-CONH2 10  39 × moreAc-V4Igl/H9A −OH Ac-ICV(2- Igl )QDWG A HRCT-COOH 11  37 × moreAc-V4Dht/H9A −OH Ac-ICV Dht QDWG A HRCT-COOH 12   5 × moreAc-V4(Bpa)/H9A −OH Ac-ICV( Bpa )QDWG A HRCT-COOH 13  49 × more+G/V4W/H9A +AN −OH H- G ICV W QDWG A HRCT AN -COOH 14  38 × moreB. Exemplary Analogs Described Herein +G/V4W/H9A +N -OH H- G ICV W QDWGA HRCT N -COOH 15  45 × more +G/V4(5f-l-W)/W7(5f-l-W),/H9A   H-GICV( 5f-

-W )QD( 5f-

-W )G A HRCTN-COOH 16 112 × more +N -OH +GN4(6f-l-W)/W7(6f-l-W)/H9A +N H-GICV( 6f-

-W )QD( 6f-

-W )G A HRCTN-COOH 17 126 × more -OH Ac-V4(5f-l-W)/H9A Ac-ICV( 5f-

-W )QDWG A HRCT-CONH₂ 18  31 × more Ac-V4W/W7(5f-l-W)/H9A Ac-ICVWQD( 5f-

-W )G A HRCT-CONH₂ 19 121 × more Ac-V4(5f-l-W)W7(5f-l-W)/H9A Ac-ICV( 5f-

-W )QD( 5f-

-W )G A HRCT-CONH₂ 20 161 × more Ac-V4(5-methoxy-W)/H9A Ac-ICV( 5-methoxy - W )QDWG A HRCT-CONH₂ 21  76 × more Ac-V4(5-methyl-W)/H9A Ac-ICV(5-meth y l-W )QDWG A HRCT-CONH₂ 22  67 × more Ac-V4(1-methyl-W)1H9AAc-ICV( 5-methyl-W )QDWG A HRCT-CONH₂ 23 264 × more Ac-V4(1-methyl-W)Ac-ICV( 1-methyl-W )QDWG A GRCT-COHN₂ Ac-V4(1-methyl-W)W7(5f-l-W)/H9AAc-ICV( 1-methyl-W )QD( 5f-

-W )G A HRCT- 24 264 × more CONH₂ Ac-V4(1-formyl-W)/H9A Ac-ICV(1-formyl-W )QDWG A HRCT-CONH₂ 25 264 × more Abbreviations used in thistable are as follows: dT = D-threonine 2-Nal = 2-napthylalanine 1-Nal =1-napthylalanine 2-1gl = 2-indanylglycine Dht = dihydrotryptophan Bpa =4-benzoyl-L-phenylalanine 5f-l-W = 5-fluoro-l-tryptophan 6f-l-W =6-fluoro-l-tryptophan 5-OH-W = 5-hydroxytryptophan 5-methoxy-W =5-methoxytryptophan 5-methyl-W = 5-methyltryptophan 1-methyl-W =1-methyltryptophan 1-formyl-W = 1-formyltryptophan

Modifications at the N-Terminus.

Acetylation of the N-terminus typically increases thecomplement-inhibiting activity of compstatin and its analogs, as can beseen specifically by comparing SEQ ID NO: 1 with SEQ ID NO:2.Accordingly, addition of an acyl group at the amino terminus of thepeptide, including but not limited to N-acetylation, is one preferredembodiment of the invention, of particular utility when the peptides areprepared synthetically. However, it is sometimes of advantage to preparethe peptides by expression of a peptide-encoding nucleic acid moleculein a prokaryotic or eukaryotic expression system, or by in vitrotranscription and translation. For these embodiments, thenaturally-occurring N-terminus may be utilized. One example of acompstatin analog suitable for expression in vitro or in vivo isrepresented by SEQ ID NOS:15-17, wherein the acetyl group is replaced byunmodified glycine at the N-terminus. SEQ ID NOS:15-17, whichadditionally comprise modifications within the peptides and at theC-termini as discussed below, are between about 45- and about 125-foldmore active than compstatin in the complement inhibition assay describedherein.

Modification within the Peptide.

Using computational methods that the rank low lying energy sequences, itwas previously determined that Tyr and Val were the most likelycandidates at position 4 to support stability and activity of thepeptide (Klepeis J L et al., 2003). It was disclosed in WO2004/026328that Trp at position 4, especially combined with Ala at position 9,yields many-fold greater activity than that of the parent peptide (forexample, compare activities of SEQ ID NOS: 4, 5 and 6 with those of SEQID NOS: 2 and 3). WO2004/026326 also disclosed that peptides comprisingthe tryptophan analogs 2-napthylalanine (SEQ ID NOS: 7, 8),1-naphthylalanine (SEQ ID NO: 9), 2-indanylglycine (SEQ ID NOS: 10, 11)or dihydrotryptophan (SEQ ID NO: 12) at position 4 were all found topossess increased complement-inhibitory activity, ranging from 5-fold to99-fold greater than compstatin. In addition, a peptide comprising thephenylalanine analog, 4-benzoyl-L-alanine, at position 4 (SEQ ID NO: 13)possessed 49-fold greater activity that did compstatin.

In accordance with the present invention, peptides comprising5-fluoro-l-tryptophan (SEQ ID NO:19) or either 5-methoxy-, 5-methyl- or1-methyl-tryptophan, or 1-formyl-tryptophan (SEQ ID NOS: 21, 22, 23 and25, respectively) at position 4 possess 31-264-fold greater activitythan does compstatin. Incorporation of 1-methyl- or 1-formyl-tryptophanincreased the activity and the binding affinity the most in comparisonto other analogs. It is believed that an indole ‘N’-mediated hydrogenbond is not necessary at position 4 for the binding and activity ofcompstatin. The absence of this hydrogen bond or reduction of the polarcharacter by replacing hydrogen with lower alkyl, alkanoyl or indolenitrogen at position 4 enhances the binding and activity of compstatin.Without intending to be limited to any particular theory or mechanism ofaction, it is believed that a hydrophobic interaction or effect atposition 4 strengthens the interaction of compstatin with C3.Accordingly, modifications of Trp at position 4 (e.g., altering thestructure of the side chain according to methods well known in the art),or substitutions of Trp analogs that maintain or enhance theaforementioned hydrophobic interaction are contemplated in the presentinvention to produce analogs of compstatin with even greater activity.Such analogs are well known in the art and include, but are not limitedto the analogs exemplified herein, as well as unsubstituted oralternatively substituted derivatives thereof. Examples of suitableanalogs may be found by reference to the following publications, andmany others: Beene, et al. (2002) Biochemistry 41: 10262-10269(describing, inter alia, singly- and multiply-halogenated Trp analogs);Babitzky & Yanofsky (1995) J. Biol. Chem. 270: 12452-12456 (describing,inter alia, methylated and halogenated Trp and other Trp and indoleanalogs); and U.S. Pat. Nos. 6,214,790, 6,169,057, 5,776,970, 4,870,097,4,576,750 and 4,299,838. Trp analogs may be introduced into thecompstatin peptide by in vitro or in vivo expression, or by peptidesynthesis, as known in the art and described in greater detail in theexamples.

In certain embodiments, Trp at position 4 of compstatin is replaced withan analog comprising a 1-alkyl substituent, more particularly a loweralkyl (e.g., C₁-C₅) substituent as defined above. These include, but arenot limited to, N(α) methyl tryptophan and 5-methyltryptophan. In otherembodiments, Trp at position 4 of compstatin is replaced with an analogcomprising a 1-alkanoyl substituent, more particularly a lower alkanoyl(e.g., C₁-C₅) substituent as defined above. In addition to exemplifiedanalogs, these include but are not limited to 1-acetyl-L-tryptophan andL-β-homotryptophan.

Thermodynamic experiments showed that incorporation of5-fluoro-l-tryptophan at position 7 in compstatin increased enthalpy ofthe interaction between compstatin and C3, relative to wildtypecompstatin, whereas incorporation of 5-fluoro-tryptophan at position 4in compstatin decreased the enthalpy of this interaction. Withoutintending to be bound to any particular mechanism, the former resultsindicate that replacement of indole hydrogens with a fluorine atom on aTrp residue at position 7 of compstatin can strengthen hydrogen bondingpotential of the indole ring, introduce new hydrogen bonding potential,or mediate an interaction with C3 through a water molecule at thebinding interface. (Katragadda M et al., 2004). Hence, modifications ofTrp at position 7 (e.g., altering the structure of the side chainaccording to methods well known in the art), or substitutions of Trpanalogs that maintain or enhance the aforementioned hydrogen bondingpotential, or mediate an interaction with C3 through a water molecule atthe binding interface, are contemplated in the present invention toproduce analogs with even greater activity. In certain embodiments, Tipanalogs whose indole rings have modifications that result in increasedhydrogen bonding potential or mediate an interaction with C3 through awater molecule at the binding interface may be introduced into position7 of the compstatin peptide by in vitro or in vivo expression, or bypeptide synthesis. A peptide comprising the tryptophan analog5-fluoro-tryptophan (SEQ ID NO:19) at position 7 was found to possess a121-fold increased activity as compared with compstatin.

In another embodiment, Tip analogs are incorporated at both positions 4and 7 of the compstatin molecule, and His at position 9 of compstatin isoptionally replaced by Ala. Thermodynamic experiments showed thatincorporation of 5-fluoro-tryptophan at positions 4 and 7 in compstatinincreased enthalpy of the interaction between compstatin and C3,relative to wildtype compstatin. Accordingly, modifications of Trp atpositions 4 and 7 (e.g., altering the structure of the side chainaccording to methods well known in the art), or substitutions of Trpanalogs that maintain or enhance the aforementioned hydrophobicinteraction with C3 via position 4 and maintain or enhance theaforementioned hydrogen bonding potential with C3 via position 7, orinteraction with C3 through a water molecule at the binding interfacevia position 7, are contemplated in the present invention to producecompstatin analogs with even greater activity. Such modified Trp or Trpanalogs may be introduced into the compstatin peptide at positions 4 and7 by in vitro or in vivo expression, or by peptide synthesis. Peptidescomprising tryptophan analogs 5-fluoro-tryptophan (SEQ. ID. NO:16) andcomprising tryptophan analogs 6-fluoro-tryptophan (SEQ. ID. NO: 17) atpositions 4 and 7 were found to possess significantly increased activityover compstatin, ranging from a 112- to a 264-fold increase in activity.In addition, peptides comprising the tryptophan analog1-methyl-tryptophan at position 4 and 5-fluoro-tryptophan at position 7(SEQ ID NO: 24) were found to possess a 264-fold increase in activityrelative to compstatin.

Modifications at the Carboxy Terminus.

Peptides produced by synthetic methods are commonly modified at thecarboxy terminus to comprise an amide instead of an acid; this commonmodification can be seen in Table 1 in compstatin (SEQ ID NO:1) andseveral analogs. Indeed, in some instances, it has been determined thatthe terminal amide-containing peptides possess greater activity than dothe terminal acid-containing peptides (compare, for example, SEQ ID NOS:5 and 7 with SEQ ID NOS: 4 and 8, respectively). Accordingly, onepreferred embodiment of the invention utilizes the C-terminal amidemodification. However, some circumstances favor the use of an acid atthe C-terminus. Such circumstances include, but are not limited tosolubility considerations and the expression of the peptides in vitro orin vivo from peptide-encoding nucleic acid molecules.

The carboxy-terminal residue of compstatin is threonine. In someembodiments of the present invention, the C-terminal threonine isreplaced by one or more naturally-occurring amino acids or analogs. Forexample, the peptide having SEQ ID NO:6 comprises D-threonine instead ofL-threonine, and further possesses a COOH group at the C-terminus. Thispeptide shows activity equal to that of peptide SEQ ID NO:5, comprisingL-threonine and CONH₂ at the C-terminus. Further, Ile has beensubstituted for Thr at position 13, to obtain a peptide with 21-foldgreater activity than that of compstatin. In addition, the peptides ofSEQ ID NOS: 14-17, which comprise a C-terminal peptide extension of Asn,or a dipeptide extension of Ala-Asn, along with a COOH at the C-terminusand a non-acetylated N-terminus, demonstrate between 38- and 126-foldgreater activity than does compstatin. They are also suitable forproduction via a prokaryotic or eukaryotic expression system, asdescribed in greater detail below.

The compstatin analogs of the present invention may be prepared byvarious synthetic methods of peptide synthesis via condensation of oneor more amino acid residues, in accordance with conventional peptidesynthesis methods. For example, peptides are synthesized according tostandard solid-phase methodologies, such as may be performed on anApplied Biosystems Model 431A peptide synthesizer (Applied Biosystems,Foster City, Calif.), according to manufacturer's instructions. Othermethods of synthesizing peptides or peptidomimetics, either by solidphase methodologies or in liquid phase, are well known to those skilledin the art. During the course of peptide synthesis, branched chain aminoand carboxyl groups may be protected/deprotected as needed, usingcommonly-known protecting groups. An example of a suitable peptidesynthetic method is set forth in Example 3. Modification utilizingalternative protecting groups for peptides and peptide derivatives willbe apparent to those of skill in the art.

Alternatively, certain peptides of the invention may be produced byexpression in a suitable prokaryotic or eukaryotic system. For example,a DNA construct may be inserted into a plasmid vector adapted forexpression in a bacterial cell (such as E. coli) or a yeast cell (suchas Saccharomyces cerevisiae), or into a baculovirus vector forexpression in an insect cell or a viral vector for expression in amammalian cell. Such vectors comprise the regulatory elements necessaryfor expression of the DNA in the host cell, positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The peptides of SEQ ID NOS:14-17, and others similarly designed, aresuitable for production by expression of a nucleic acid molecule invitro or in vivo. A DNA construct encoding a concatemer of the peptides,the upper limit of the concatemer being dependent on the expressionsystem utilized, may be introduced into an in vivo expression system.After the concatemer is produced, cleavage between the C-terminal Asnand the following N-terminal G is accomplished by exposure of thepolypeptide to hydrazine.

The peptides produced by gene expression in a recombinant procaryotic oreucaryotic system may be purified according to methods known in the art.Examples 1 and 2 set forth methods suitable for use in the presentinvention. In one embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant peptideis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium.

A combination of gene expression and synthetic methods may also beutilized to produce compstatin analogs. For example, an analog can beproduced by gene expression and thereafter subjected to one or morepost-translational synthetic processes, e.g., to modify the N- orC-terminus or to cyclize the molecule.

The structure of compstatin is known in the art, and the structures ofthe foregoing analogs are determined by similar means. Once a particulardesired conformation of a short peptide has been ascertained, methodsfor designing a peptide or peptidomimetic to fit that conformation arewell known in the art. See, e.g., G. R. Marshall (1993), Tetrahedron,49: 3547-3558; Hruby and Nikiforovich (1991), in Molecular Conformationand Biological Interactions, P. Balaram & S. Ramasehan, eds., IndianAcad. of Sci., Bangalore, PP. 429-455). Of particular relevance to thepresent invention, the design of peptide analogs may be further refinedby considering the contribution of various side chains of amino acidresidues, as discussed above (i.e., for the effect of functional groupsor for steric considerations).

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for the purpose of providing thespecific backbone conformation and side chain functionalities requiredfor binding to C3 and inhibiting complement activation. Accordingly, itis contemplated as being within the scope of the present invention toproduce C3-binding, complement-inhibiting compounds through the use ofeither naturally-occurring amino acids, amino acid derivatives, analogsor non-amino acid molecules capable of being joined to form theappropriate backbone conformation. A non-peptide analog, or an analogcomprising peptide and non-peptide components, is sometimes referred toherein as a “peptidomimetic” or “isosteric mimetic,” to designatesubstitutions or derivations of the peptides of the invention, whichpossess the same backbone conformational features and/or otherfunctionalities, so as to be sufficiently similar to the exemplifiedpeptides to inhibit complement activation.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art (see, e.g., Zhao B et al., 1995;Beeley, N. 1994; and, Hruby, V J 1993) Assuming rotational constraintssimilar to those of amino acid residues within a peptide, analogscomprising non-amino acid moieties may be analyzed, and theirconformational motifs verified, by means of the Ramachandran plot (Hruby& Nikiforovich 1991), among other known techniques.

The compstatin analogs of the present invention can be modified by theaddition of polyethylene glycol (PEG) components to the peptide. As iswell known in the art, PEGylation can increase the half-life oftherapeutic peptides and proteins in vivo. In one embodiment, the PEGhas an average molecular weight of about 1,000 to about 50,000. Inanother embodiment, the PEG has an average molecular weight of about1,000 to about 20,000. In another embodiment, the PEG has an averagemolecular weight of about 1,000 to about 10,000. In an exemplaryembodiment, the PEG has an average molecular weight of about 5,000. Thepolyethylene glycol may be a branched or straight chain, and preferablyis a straight chain.

The compstatin analogs of the present invention can be covalently bondedto PEG via a linking group. Such methods are well known in the art.(Reviewed in Kozlowski A. et al. 2001; see also, Harris J M and ZalipskyS, eds. Poly(ethylene glycol), Chemistry and Biological Applications,ACS Symposium Series 680 (1997)). Non-limiting examples of acceptablelinking groups include an ester group, an amide group, an imide group, acarbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, asuccinimide group (including without limitation, succinimidyl succinate(SS), succinimidyl propionate (SPA), succinimidyl carboxymethylate(SCM), succinimidyl succinamide (SSA) and N-hydroxy succinimide (NHS)),an epoxide group, an oxycarbonylimidazole group (including withoutlimitation, carbonyldimidazole (CDI)), a nitro phenyl group (includingwithout limitation, nitrophenyl carbonate (NPC) or trichlorophenylcarbonate (TPC)), a trysylate group, an aldehyde group, an isocyanategroup, a vinylsulfone group, a tyrosine group, a cysteine group, ahistidine group or a primary amine. In certain embodiments, the linkinggroup is a succinimide group. In one embodiment, the linking group isNHS.

The compstatin analogs of the present invention can alternatively becoupled directly to PEG (i.e., without a linking group) through an aminogroup, a sulfhydral group, a hydroxyl group or a carboxyl group. In oneembodiment, PEG is coupled to a lysine residue added to the C-terminusof compstatin.

PEGylation is one way to increase in vivo retention of therapeuticpeptides and proteins. The in vivo clearance of peptides can also bereduced by linking the peptides to certain other peptides. For instance,certain albumin binding peptides display an unusually long half-life of2.3 h when injected by intravenous bolus into rabbits (Dennis et al.,2002). A peptide of this type, fused to the anti-tissue factor Fab ofD3H44 enabled the Fab to bind albumin while retaining the ability of theFab to bind tissue factor (Nguyen et al., 2006). This interaction withalbumin resulted in significantly reduced in vivo clearance and extendedhalf-life in mice and rabbits, when compared with the wild-typeD3H44Fab, comparable with those seen for PEGylated Fab molecules,immunoadhesins, and albumin fusions. As described in Example 11 herein,the inventors have synthesized a compstatin analog fused with analbumin-binding peptide and demonstrated that the fusion protein isactive in inhibiting complement activation.

The complement activation-inhibiting activity of compstatin analogs,peptidomimetics and conjugates may be tested by a variety of assaysknown in the art. In a preferred embodiment, the assay described inExample 4 is utilized. A non-exhaustive list of other assays is setforth in U.S. Pat. No. 6,319,897, including, but not limited to, (1)peptide binding to C3 and C3 fragments; (2) various hemolytic assays;(3) measurement of C3 convertase-mediated cleavage of C3; and (4)measurement of Factor B cleavage by Factor D.

The peptides and peptidomimetics described herein are of practicalutility for any purpose for which compstatin itself is utilized, asknown in the art. Such uses include, but are not limited to: (1)inhibiting complement activation in the serum, tissues or organs of apatient (human or animal), which can facilitate treatment of certaindiseases or conditions, including but not limited to but not limited to,age-related macular degeneration, rheumatoid arthritis, spinal cordinjury, Parkinson's disease, and Alzheimer's disease; (2) inhibitingcomplement activation that occurs during use of artificial organs orimplants (e.g., by coating or otherwise treating the artificial organ orimplant with a peptide of the invention); (3) inhibiting complementactivation that occurs during extracorporeal shunting of physiologicalfluids (blood, urine) (e.g., by coating the tubing through which thefluids are shunted with a peptide of the invention); and (4) inscreening of small molecule libraries to identify other inhibitors ofcompstatin activation (e.g., liquid- or solid-phase high-throughputassays designed to measure the ability of a test compound to competewith a compstatin analog for binding with C3 or a C3 fragment).

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.The materials and methods set forth in Examples 1-5 were utilized togenerate the results described in Examples 6-11.

Example 1 Bacterial Expression of Compstatin

A compstatin analog with the following sequence, NH₂-GICVWQDWGAHRCTN-OH(“G(-1)/V4W/H9A/N14”) (SEQ ID NO:15) was expressed in fusion with chitinbinding domain and the DnaB intein (New England Biolabs, Beverly,Mass.). Guided by the peptide sequence and the codon usage for E. colithe following genetic code was used to generate a synthetic gene forthis peptide with the following sequence:

(SEQ ID NO: 29) ^(5′)ATTTGCGTTTGGCAGGATTGGGGTGCGCACCGTTGCACCAATTAA^(3′)

To clone the synthetic gene into the pGEM-T vector, a 5′ flanking regioncontaining a SapI site and 3′ flanking region containing a PstI sitewere designed. To construct the synthetic gene, the four overlappingoligonucleotides shown below were designed using DnaWorks software andsynthesized at Invitrogen Inc. (Carlsbad, Calif.):

(SEQ ID NO: 30) ^(5′)GGTGGTGCTCTTCCAACGGTATTTGCGTTTGGCAGGA^(3′)(SEQ ID NO: 31) ^(5′)TTGGGGTGCGCACCGTTGCACCAATTAACTGCAGG^(3′)(SEQ ID NO: 32) ^(3′)CAACGTGGTTAATTGACGTCCGC^(5′) (SEQ ID NO: 33)^(3′)CATAAACGCAAACCGTCCTAACCCCACGCGTGG^(5′)

The overlapping DNA fragments were assembled by PCR as described byStemmer et al., 1995. The resulting gene was amplified using thefollowing primers:

^(5′)CGCCTGCAGTTAATTGGT^(3′) (SEQ ID NO: 34)^(5′)GGTGGTGCTCTTCCAACG^(3′) (SEQ ID NO: 35)

The PCR-amplified fragments of compstatin were then cloned into thepGEM-T vector, and the resulting clone was digested with PstI and SapI.The PstI-SapI fragment encoding the compstatin analog was furthersubcloned into the expression vector pTWIN1, which had been predigestedwith PstI and SapI; the sequence of the clone was verified by DNAsequencing.

To express the compstatin analog, ER2566 E. coli cells transformed withthe compstatin clone were grown in SOB medium (20 g/L tryptone, 5 g/Lyeast extract, 0.5 g/L NaCl, 2.5 mM KCl, 10 mM MgCl₂) at 37° C. When anOD₆₀₀ 0.7 was reached, expression was induced by the addition of IPTG toa final concentration of 0.3 mM, followed by an additional incubation at37° C. for 4 hr. Cells were collected by centrifugation and lysed bysonication in buffer B1 (20 mM phosphate buffer, pH 8.5, with 500 mMNaCl and 1 mM EDTA) supplemented with 0.2% Tween-20. The cell extractwas centrifuged, and the soluble fraction was applied to a chitinbinding column (New England Biolabs, Beverly, Mass.) pre-equilibratedwith buffer B1. The column was washed with 100 ml of buffer B1, followedby a quick wash with 3 column volumes of buffer B2 (50 mM ammoniumacetate, pH 7.0). The column was incubated at room temperature for 20hr, and the peptide was eluted with Buffer B2, lyophilized and furtherpurified on a C18 HPLC column. The purified peptide was identified usingMALDI-TOF mass spectrometry.

Example 2 Expression of Tryptophan Analogs of Compstatin in E. coli

To express compstatin analogs containing tryptophan derivatives, thepTWIN1-compstatin clone was transformed into the ER2566 Trp 82auxotroph. Expression was carried out in M9 minimal medium supplementedwith 1 mM L-tryptophan as described above. Cells were grown to an OD₆₀₀0.8-1.0, then collected by centrifugation and resuspended in freshminimal medium containing 2 mM of the desired tryptophan analog(s):5-fluoro-tryptophan, 6-fluoro-tryptophan, 7-aza-tryptophan or5-hydroxy-tryptophan. The expressed compstatin analogs were furtherpurified as described in Example 1.

Example 3 Peptide Synthesis

Peptide synthesis and purification was performed as described by Sahu etal., 1996; Sahu et al., 2000; and Mallik et al., 2005. Briefly, peptideswere synthesized in an Applied Biosystem peptide synthesizer (model431A) using Fmoc amide resin and standard side chain protecting groups.Peptides were cleaved from the resin by incubation for 3 hours at 22° C.with a solvent mixture containing 5% phenol, 5% thioanisole, 5% water,2.5% ethanedithiol, and 82.5% trifluoroacetic acid (TFA). The reactionmixture was filtered through a fitted funnel, precipitated with coldether, dissolved in 50% acetonitrile containing 0.1% TFA, andlyophilized.

The crude peptides obtained after cleavage were dissolved in 10%acetonitrile containing 0.1% TFA and purified using a reverse phase C-18column (Waters, Milford, Mass.). Disulfide oxidation was achieved by anon-resin cyclization method using the reagent Thallium (III)trifluoroacetate. This method eliminates the dilute solution oxidationsteps and subsequent time-consuming concentration through lyophilizationsteps prior to reverse-phase HPLC. Using this method, the multimerformation was nonexistent and a high level (˜90%) of fully deprotected,oxidized or cyclized material was obtained. The identity and purity ofall peptides were confirmed by laser desorption mass spectroscopy andHPLC.

For the synthesis of the 5-fluoro-tryptophan, 1-methyl-tryptophan, and5-methyl-tryptophan analogs, Fmoc-dl-derivatives were used. Separationof the enantiomeric peptides was performed as described by Meyers et al.1978. The dl mixture of each peptide was separated into d and l isomericpeptides on a C18 reversed-phase HPLC column using 10% acetonitrile in0.01M ammonium acetate, pH 4.1. The isomeric identity of the elutedpeptides was determined by treating the peptides with V8 protease,followed by analysis using MALDI-TOF mass spectrometry (MicroMassTOFspec2E).

Example 4 Complement Inhibition Assays

Inhibitory activity of compstatin and its analogs on the complementsystem was determined by measuring their effect on the activation of thecomplement system by immunocomplexes. Complement activation inhibitionwas assessed by measuring the inhibition of C3 fixation toovalbumin-anti-ovalbumin complexes in normal human plasma. Microtiterwells were coated with 50 μl of ovalbumin (10 mg/ml) for 2 hr at 25° C.(overnight at 4° C.). The wells were saturated with 200 μl of 10 mg/mlBSA for 1 hr at 25° C. and then a rabbit anti-ovalbumin antibody wasadded to form an immunocomplex by which complement can be activated.Thirty microliters of peptides at various concentrations were addeddirectly to each well followed by 30 μl of a 1:80 dilution of humanplasma. After 30 min incubation, bound C3b/iC3b was detected using agoat anti-human C3 HRP-conjugated antibody. Color was developed byadding ABTS peroxidase substrate and optical density measured at 405 nm.

The absorbance data obtained at 405 nm were translated into % inhibitionbased on the absorbance corresponding to 100% complement activation. The% inhibition was plotted against the peptide concentration, and theresulting data set was fit to the logistic dose-response function usingOrigin 7.0 software. The concentration of the peptide causing 50%inhibition of C3b/iC3b deposition was taken as the IC₅₀ and used tocompare the activities of various peptides. IC₅₀ values were obtainedfrom the fitted parameters that achieved the lowest chi-square value.

Example 5 Isothermal Titration Calorimetry Analysis of the InteractionC3 with Compstatin and its Analogs

Isothermal titration calorimetry experiments were performed using theMicrocal VP-ITC calorimeter (Microcal Inc, Northampton, Mass.). Proteinconcentrations of 3.5-5 μM and peptide concentrations of 80-200 μM wereused for these experiments. All titrations were performed in PBS (10 mMphosphate buffer with 150 mM NaCl, pH 7.4). In each experiment, thetarget protein, C3, was loaded into the cell, and peptide was loadedinto the syringe. All experiments were performed at 25° C. and for eachexperiment, 2-μl peptide injections were made into the cell containingthe protein. In each experiment, the raw isotherms were corrected forthe heats of dilution by subtracting the isotherms representing peptideinjections into the buffer. The resulting isotherms were fit to variousmodels within the Origin 7.0 software, and the model that achieved thelowest chi square value was deemed to be appropriate for the respectivedataset. Binding affinity and entropy values were plotted against log Pvalues.

Example 6 Role of Tryptophan in C3-Compstatin Interaction as Assessed byBacterially Expressed Compstatin Analogs

Four different tryptophan analogs that differ in the chemical nature ofthe indole ring were incorporated into compstatin using anintein-mediated protein expression system. Following expression, thepeptides were purified in a single step with a final yield of 2 mg/L ofculture. The tryptophan analogs 5-fluoro-tryptophan,6-fluoro-tryptophan, 7-aza-tryptophan and 5-hydroxy-tryptophan were alsoexpressed using the ER2566/Trp 82 auxotroph as indicated by the MALDIprofiles, and the resulting peptides were purified to homogeneity.Native compstatin and analogs were cyclized in vivo through a disulfidebond, as evidenced by their inability to react with PHMB. All peptideswere further purified on a reverse-phase C18 HPLC column.

The activity of the expressed compstatin analog G(-1)/V4W/H9A/N14 (SEQID NO:15) exhibited an IC₅₀ of 1.2 μM, which is similar to the activityobserved for the Ac-V4W/H9A analog (SEQ ID NO:5). This finding indicatesthat the glycine located at the N-terminus of the expressed peptideplays a role similar to that of the acetyl group located at theN-terminus of the Ac-V4W/H9A analog.

All the expressed compstatin analogs except the 7-aza tryptophan analogwere found to be active at the concentrations tested. However, thepeptide showed different levels of activity relative to the analog,Ac-V4W/H9A (FIG. 1; Table 2). Compstatin containing 6-fluoro-tryptophanand 5-fluoro-tryptophan as well as alanine at position 9 exhibited a 2.8and 2.5-fold higher activity, respectively, than that of the Ac-V4W/H9Aanalog.

TABLE 2 Complement inhibitory activity of the expressed peptides SEQ IDRelative Expressed peptide NO: IC₅₀ (μM) activity* Ac-V4W/H9A^(b) 5 1.245 G(−1)/V4W/H9A/N14 15 1.2 45 G(−1)/V4(5fW)/W7(5fW)/H9A/N14 16 0.48 112G(−1)/V4(6fW)/W7(6fW)/H9A/N14 17 0.43 126 G(−1)/V4(5-OH^(a)-W)/W7(5-OH-27 33 1.6 W)/H9A/N14 G(−1)/V4(7-aza-W)/W7(7-aza- 28 122 0.44 W)/H9A/N14*relative to the activity of the peptide H-I(CVVQDWGHHRC)T-NH₂(compstatin, SEQ ID NO: 1) ^(c)represents hydroxy ^(b)synthetic peptide

Without being limited to any particular mechanism, it is believed thatadding fluorine atom increases the activity of the peptide by increasingthe hydrophobicity of the indole ring. The incorporation of lesshydrophobic tryptophan analogs 5-hydroxy tryptophan and 7-aza-tryptophanwas also investigated. In contrast to the results with the 5-fluoro and6-fluoro analogs, compstatin analogs containing 5-hydroxy-tryptophanshowed 27.5-fold loss in the activity compared to the Ac-V4W/H9A analog(SEQ ID NO:5), and the peptide containing 7-aza-tryptophan showed noactivity at all at the concentrations tested. 7-aza-tryptophan resemblestryptophan in molecular structure except that it has a nitrogen atom atposition 7 of the indole ring as opposed to a carbon atom. The loss inactivity observed upon substitution of 7-aza-tryptophan shows therelative importance of this carbon atom.

Example 7 Role of Individual Tryptophans in C3-Compstatin Interaction

Solid-phase peptide synthesis was used to generate compstatin analogswith 5-fluoro-tryprophan incorporated selectively at position 4,position 7, or both positions 4 and 7, with alanine at position 9.Synthesis was undertaken using Fmoc-5-fluoro-dl-tryptophan. Thisreaction yielded an enantiomeric mixture of the peptides bearing5-fluoro-d-tryptophan and 5-fluoro-l-tryptophan. Three differentpeptides were synthesized: two peptides with single substitutionindependently at position 4 or 7 and one peptide with substitutions atboth positions 4 and 7. While a mixture of 5-fluoro-l-tryptophan and5-fluoro-d-tryptophan analogs could occur in the case of the singlesubstitutions, a mixture of four enantiomeric combinations was possiblein the case of the double substitution. Each of the peptide mixtures wasfurther subjected to reversed-phase HPLC to separate the peptideenantiomers. Identification of the enantiomers was carried out bydigesting the peptides with V8 protease and subsequently analyzing thedigested product using MALDI. V8 protease cleaves at the C-terminal sideof Asp residues only when followed by an l-amino acid. Identification ofcleavage products in the mass spectra indicated that the l-enantiomericpeptide eluted first followed by the d-form, where no cleavage fragmentswere detected.

All the peptides, containing either 5-fluoro-l-tryptophan or5-fluoro-d-tryptophan or both, were tested for their complementinhibitory activity. The synthetic peptide substituted with5-fluoro-l-tryptophan in both the positions showed a 2.5-fold higheractivity than that of Ac-V4W/H9A (SEQ ID NO:5) (Table 3).

TABLE 3 Complement inhibitory activity of the synthetic compstatinanalogs containing 5-fluoro-1-tryptophan SEQ ID Relative Peptide NO:IC₅₀ (μM) activity* Ac-V4W/H9A 5 1.20 45 Ac-V4(5f-l-W)/H9A 18 1.74 31Ac-V4W/W7(5f-l-W)/H9A 19 0.446 121 Ac-V4(5f-l-W)/W7(5f-l- 20 0.482 112W)/H9A *relative to the activity of the peptide H-I(CVVQDWGHHRC)T-NH₂(compstatin, SEQ ID NO: 1)

Complement inhibition assays (FIG. 2; Table 3) indicated that (a)substitution of 5-fluoro-l-tryptophan at position 4 alone rendered thepeptide at least 1.5 times less active than Ac-V4W/H9A (SEQ ID NO:5).Substitution of 5-fluoro-l-tryptophan at position 7 alone increased theactivity 2.7-fold when compared to Ac-V4W/H19A. Substitution of5-fluoro-l-tryptophan simultaneously at positions 4 and 7 also yielded a2.5-fold increase in the activity relative to Ac-V4W/H9A (SEQ ID NO:5).Substitution of 5-fluoro-d-tryptophan at either position 4 or 7, orboth, rendered the peptide inactive.

Example 8 Thermodynamic Basis for the Tryptophan-Mediated Recognition ofCompstatin by C3

Isothermal titration calorimetry was used to examine the binding of thepeptides to C3 and investigate the thermodynamic basis for theiractivities. The calorimetric data obtained for the interaction of allthe peptides with C3 fit to a one set of sites model with stoichiometryclose to 1. It is believed that the binding of these peptides to C3occurs in a 1:1 ratio. The thermodynamic parameters resulting from thesefits are shown in Table 4. As evident from the K_(d) values, the peptidewith a single substitution of 5-fluoro-l-tryptophan at position 7 and adouble substitution at positions 4 and 7 exhibited tighter binding thanthe Ac-V4W/H9A (SEQ ID NO:5) and the Ac-V4(5f-l-W)/H9A (SEQ ID NO:18)analogs. This finding is in agreement with the relative activitiesobserved in the complement inhibition assay (Table 3), indicating that abinding-activity correlation exists.

All peptides bound to C3 with a negative enthalpy and positive entropy.Such binding is a characteristic of the interaction of compstatin withC3. Among all the peptides examined, the position 7-substitutedAc-V4W/W7(5f-l-W)/H9A analog (SEQ ID NO:19) exhibited a higher bindingenthalpy (ΔH=−21.83, ΔΔH=−3.69) than did its wild-type counterpart. Theposition 4-substituted Ac-V4(5f-l-W)/H9A analog (SEQ ID NO:18) bound toC3 with an enthalpy of −16.69 kcal/mole, 1.45 kcal/mole lower than thatexhibited by its wild-type counterpart.

Incorporation of 5-fluoro-tryptophan at position 4 led to a loss inenthalpy of 1.45 kcal/mole relative to that of tryptophan at thisposition (Table 4). Since the only difference between tryptophan and5-fluoro-tryptophan is the fluorine atom at C5 of the indole, this lossin enthalpy can be attributed to the replacement of hydrogen withfluorine.

TABLE 4 Thermodynamic parameters for the interaction of syntheticcompstatin analogs containing 5-fluoro-l-tryptophan and C3 SEQ ID K_(d)(kcal/mole) peptide NO: (μM) ΔH ΔΔH −TΔS −TΔΔS ΔG ΔΔG Ac-V4W/H9A 5 0.14−18.14 0 8.79 0 −9.4 0 Ac-V4(5f-l-W)/H9A 18 0.15 −16.69 1.45 7.39 −1.4−9.4 0 Ac-V4W/W7(5f-l- 19 0.035 −21.83 −3.69 11.56 2.77 −10.25 −1 W)/H9AAc-V4(5f-l-W)/W7(5f-l- 20 0.017 −17.33 0.81 6.73 −2.06 −10.6 −1.2 W)/H9A

Incorporation of 5-fluoro-tryptophan at position 7 increased theenthalpy by 3.69 kcal/mole relative to wild-type (Table 4). Withoutbeing limited to any particular mechanism, it is believed thattryptophan at position 7 is participating in an enthalpically favorableinteraction such as hydrogen bonding. Replacing one of the indolehydrogens with a fluorine atom might strengthen the hydrogen bondingcharacter of the indole NH due to the drop in pK_(a). Alternatively, thefluorine forms a hydrogen bond as a result of its electron-donatingnature, as has been demonstrated in the structure of the tetradeca(3-fluorotyrosyl)glutathione transferase.

Another explanation for the observed increase in enthalpy is that awater molecule is bridging the interaction between the fluorine atom anda hydrogen acceptor on C3, in which case two hydrogen bonds (equivalentto about 4 kcal/mole energy) need to be formed. Support for this theorycomes from the decrease in entropy observed for the interaction of theposition 7-substituted Ac-V4W/W7(5fW)/H9A analog (SEQ ID NO:19) relativeto the wild-type analog (Table 4), a decrease that could be produced bythe binding of an additional water molecule at the interface.Water-mediated interactions between fluorine atoms and other hydrogenbond acceptors have been observed in other systems.

Binding of the double-substituted analog to C3 yielded an enthalpychange of −19.85 kcal/mole, an entropy change of −9.35 kcal/mole and afree energy change of −10.5 kcal/mole. It is believed that incorporationof 5-fluoro-tryptophan simultaneously at both positions abrogates theeffects of the single substitutions.

Example 9 Additional Compstatin Analogs

Incorporation of Tryptophan Analogs at Position 4.

It was shown in Examples 5 and 6 that substitution of valine withtryptophan at position 4 of compstatin increased its activity 45-fold.To further investigate the nature of interaction mediated by residue atposition 4 during the course of the binding of compstatin to C3, thetryptophan at position 4 was replaced with tryptophan analogs and2-napthylalanine.

ELISA-based assays were used to test the activity of all the peptideanalogs bearing tryptophan analogs at position 4 and alanine at position9. While substitution with 1-methyl-tryptophan (Ac-V4(1-methyl-W)/H9A)(SEQ ID NO:23) and 2-naphthylalanine (Ac-V4(2-Nal)/H9A) (SEQ ID NO:7)increased the activity over compstatin 264 and 99-fold, respectively,substitution of 5-fluoro-tryptophan (Ac-V4(5f-l-W)/W7/H9A) (SEQ ID NO:18and 5-methyl tryptophan (Ac-V4(5-methyl-W)/H9A) (SEQ ID NO:22) resultedin a lower activity; to 31 and 67-fold greater than the activityexhibited by the wild-type peptide (Table 5). FIG. 3 shows theinhibitory curves depicting the activity and Table 5 shows the IC₅₀values calculated from the curves and the relative activities of thepeptides in comparison to the activity of original compstatin. FIG. 5shows inhibitory constants (IC₅₀) plotted against log P values oftryptophan analogs and 2-napthylalanine.

TABLE 5 Complement inhibitory activity of the compstatin analogs SEQ IDRelative Peptide NO: IC₅₀ (μM) activity* Ac-V4W/H9A 5 1.20 45Ac-Y4(5f-l-W)/7W/H9A 18 1.74 31 Ac-V4W/W7(5f-/-W)/H9A 19 0.446 121Ac-V4(5f-l-W)/W7(5f-l-W)/H9A 20 0.482 112 Ac-V4W/7(5-methoxy 29 0.46 0.5W)/H9A Ac-V4(5-methoxy 21 0.71 76 W)/7W/H9A Ac-V4(5-methyl W)/7W/H9A 220.81 67 Ac-V4(1-methyl W)/7W/H9A 23 0.205 264 Ac-V4(2-Nal)/W7/H9A 70.545 99 Ac-V4(l-methyl W)/W7(5f-l- 24 0.205 264 W)/H9A *Relative to theactivity of H-I(CVVQDWGHHRC)T-NH₂ (compstatin, SEQ ID NO: 1).

The binding of compstatin peptides was also investigated usingisothermal titration calorimetry. The calorimetric data obtained for theinteraction of all the peptides with C3 fit to a one set of sites modelwith stoichiometry close to 1 (FIG. 4). This result suggests that thebinding of these peptides to C3 occurs in a 1:1 ratio. The thermodynamicparameters resulted from these fits are shown in Table 6. As evidentfrom the Kd values, Ac-V4(1-methyl-W)/H9A exhibited higher bindingaffinity (K_(d)=0.015 μM) compared to all other peptides having a singlesubstitution at position 4. Plotting these values against the log Pvalues of analogs indicates that a correlation exists between bindingaffinity and hydrophobic nature of the tryptophan analogs and2-napthylalanine. As per the correlation, binding affinity increaseswith an increase in the hydrophobicity of the analog incorporated atposition 4. This observation is consistent with the correlation shownbetween log P and the inhibitory constants.

TABLE 6 Thermodynamic parameters for the interaction of syntheticcompstatin analogs containing 5-fluoro-l-tryptophan and C3 SEQ ID K_(d)(kcal/mole) Peptide NO. (μM) ΔH ΔΔH −TΔS −TΔΔS ΔG ΔΔG Wild-type 1 0.14−18.14 0 8.79 0 −9.4 0 Ac-V4(5f-l-W/H9A 18 0.15 −16.69 1.45 7.39 −1.4−9.4 0 Ac-V4(5-methyl- 22 0.12 −17.75 0.34 8.2 −0.54 −9.55 −0.15 W)/H9AAc-V4(1-methyl- 23 0.015 −17.59 0.81 6.94 −1.85 −10.65 −1.1 W)/H9AAc-V4(2-Nal)/H9A 7 0.11 −14.27 3.87 4.8 −3.99 −9.5 −0.1 Ac-V4W/W7(5f-l-19 0.035 −21.83 −3.69 11.56 2.77 −10.25 −0.8 W)/H9A Ac-V4(1-methyl- 240.017 −17.33 0.81 6.73 −2.06 −10.6 −1.2 W)/W7(5f-l-W)/H9A

All the peptides bound to C3 with a negative enthalpy and positiveentropy, suggesting that the binding is enthalpy-driven. Such binding isa characteristic of the interaction of compstatin with C3. However, thebinding of these peptides is characterized by an enthalpy change lowerthan the wild-type, and entropy change shifted towards favorable end.FIG. 5B shows a plot of log P vs. −TΔS, which indicates that with anincrease in the hydrophobicity of the analogs incorporated at position4, the entropy is more favored thus making a positive impact on the freeenergy change.

Incorporation of Tryptophan Analogs at Position 7.

It was proposed in Example 7 that tryptophan at position 7 makes ahydrogen bond with a residue on C3. To examine this possibility further,tryptophan at position 7 was replaced with tryptophan analogs similar tothe replacements at position 4 to elucidate the nature of interactionmade by tryptophan at this position. Substitution with5-fluoro-tryptophan (Ac-V4W/W7(5f-l-W)/H9A) (Seq ID NO:19), yielded a121-fold more active peptide. (FIG. 3, Table 5). Substitutions oftryptophan 7 with the analog 5-methyl trp or 1-methyl trp renderedcompstatin inactive (data not shown). Thus, no correlation between theactivity and hydrophobicity of tryptophan analogs was observed.

The thermodynamic properties of the different Trp 7-analogs wasinvestigated in parallel by calorimetry. (Table 6). Since no binding wasdetected for peptides containing either the 5-methyl trp or 1-methyl trpat position 7, the binding parameters do not exist. Only the peptideAc-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) bound to C3. The binding affinitywas 0.035 μM, which is greater that that observed for all the peptideshaving a Trp analog at position 4, except for the peptideAc-V4(1-methyl-W)/H9A (SEQ ID NO:23). In contrast to the peptides havinga Trp analog at position 4, Ac-V4W/W7(5f-l-W)/H9A (SEQ ID NO:19) boundto C3 with high favorable binding enthalpy change (ΔH=−21.83, ΔH=−3.69)and unfavorable entropy change (−TΔS=11.56, −TΔΔS=2.77), suggestingadditional favorable non-covalent interactions of polar nature.

The results show that incorporation of 5-fluoro-tryptophan at position 7results in an increase in the activity of compstatin, whereasincorporation of analogs 5-methyl-tryptophan and 1-methyl-tryptophanrenders compstatin inactive. The loss of activity of compstatin uponincorporation of 1-methyl-tryptophan supports the conclusion that thehydrogen bond mediated by N—H of Trp 7 is important for the interactionof compstatin with C3. In addition, the complete loss of activity ofcompstatin upon incorporation of 5-methyl-tryptophan suggests that ahydrophobic amino acid is not well tolerated at position 7.

Incorporation of Tryptophan Analogs at Both Positions 4 and 7.

Since the substitution of tryptophans at position 4 with1-methyl-tryptophan and position 7 with 5-fluoro-tryptophan yieldedcompstatin analogs that showed a drastic increase in the activity, acompstatin analog containing substitutions at positions 4 and 7 wasgenerated. The resulting peptide (Ac-V4(1-methyl-W)/W7(5f-l-W)/H9A) (SEQID NO:24) generated an inhibition curve similar to that of the singlesubstitution with 1-methyl-tryptophan (Ac-V4(1-methyl-W)/H9A) (SEQ IDNO:23), (FIG. 3, Table 5). The binding affinity (K₁=0.017) observed forthis peptide in the calorimeter is also similar to that ofAc-V4(1-methyl-W)/H9A (SEQ ID NO:23). These observations suggest that5-fluoro-tryptophan has no effect at position 7 in the presence of1-methyl-tryptophan at position 4 under these experimental conditions.

Incorporation of Another Tryptophan Analog at Position 4.

To further investigate the nature of interaction mediated by residue atposition 4 during the course of the binding of compstatin to C3, thetryptophan at position 4 was replaced with the tryptophan analog1-formyl-tryptophan.

FIG. 6 shows a comparison of percent complement inhibition vs. peptideconcentration for Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23) (circles) andAc-V4(1-formyl-W)/H9A (SEQ ID NO:25). As can be seen, the 1-formyl-Wanalog was essentially identical to the 1-methyl-W analog in itscomplement inhibition activity.

Example 10 PEGylation of Compstatin Analog

A prolonged half-life of compstatin is advantageous for its use inchronic treatments. Extending the half-life of tested therapeuticpeptides has been achieved in several instances through PEGylation (seeVeronese et al., 2001), as PEG has the ability to delay the eliminationof biomolecules from the circulation through a variety of mechanisms,including decreasing renal clearance, proteolysis and immunogenicity.PEGylation involves covalent attachment of PEG polymers tomacromolecules, preferably to the primary amine of lysines.

This example describes the preparation of a PEGylated compstatin analog,Ac-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36) and evaluation of thecompound for its ability to inhibit complement activation.

Fmoc-NH-NHS-5000 PEG was purchased from Nektar transformingtherapeutics, 490 discovery Dr, Huntsville, Ala. 35806.

The compound Ac-V4(1-methyl-W)/H9A-K-PEG 5000 (SEQ ID NO:36) wassynthesized chemically by Fmoc solid-phase peptide chemistry accordingto a modified standard protocol. Briefly, PEG was dissolved in 3 ml ofdichloromethane, 1 ml of 2M DIEA was added manually, and the PEG wasmixed for 5 minutes.

Then the PEG was transferred to the vessel, and left to coupleovernight. The PEG was then deprotected with 20% piperidine for 20 min.

Then the synthesis proceeded according to the standard protocol, with alysine incorporated at the C-terminus of the molecule for the purpose oflinking the PEG to its side chain.

Final cleavages of the peptides was achieved with Reagent D(TFA:H2O:TIS:Phenol, 87.5:5:2.5:5) (4 mL) at 25 C for 90 min, to providethe desired product. The peptide was then purified on a C18reversed-phase HPLC column, lyophilized and characterized by MALDI-TOF.

The PEGylated compstatin analog was tested for complement-inhibitingactivity using the in vitro assay described in Example 4. As shown inFIG. 7, the PEGylated analog was active in inhibiting complementactivation, however, seven-fold more conjugate was required to achievethe same amount of inhibition as the non-PEGylated analog,Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23).

Example 11 Albumin Binding Protein Conjugate of Compstatin Analog

Dennis et al. (2002) identified a series of peptides having the coresequence DICLPRWGCLW (SEQ ID NO:37) that specifically bound serumalbumin from multiple species with high affinity. These peptides boundto albumin with 1:1 stoichiometry at a site distinct from known smallmolecule binding sites. The peptide SA21 (AcRLIEDICLPRWGCLWEDDNH2; SEQID NO:38) has an unusually long half-life of 2.3 h when injected byintravenous bolus into rabbits. As mentioned in the DetailedDescription, a related sequence, fused to the anti-tissue factor Fab ofD3H44 enabled the Fab to bind albumin with similar affinity to that ofSA21 while retaining the ability of the Fab to bind tissue factor1(Nguyen et al. 2006). This interaction with albumin resulted in reducedin vivo clearance of 25- and 58-fold in mice and rabbits, respectively,when compared with the wild-type D3H44Fab. The half-life was extended37-fold to 32.4 h in rabbits and 26-fold to 10.4 h in mice, achieving25-43% of the albumin half-life in these animals. These half-livesexceed those of a Fab 2 and are comparable with those seen for PEGylatedFab molecules, immunoadhesins, and albumin fusions.

This example describes the synthesis of a Compstatin analog fused withan albumin-binding peptide and its activity in in vitro assays forcomplement inhibition.

The compound 4(1MeW)-ABP was synthesized chemically by Fmoc solid-phasepeptide chemistry according to standard protocols. The N- and C-terminiof the peptide were protected with acetyl and amide groups. The peptidewas further purified on a C18 reversed-phase HPLC column, lyophilized,and characterized by MALDI mass spectrometry.

For cyclization, the peptide-resin (0.10 mmol/g loading based on aminoacid analysis) was swollen in dichloromethane (DCM) (2 mL) for 5 min,filtered and treated with 94:1:5 DCM/TFA/TIS (5 mL) at 25° C. 3 times×2min each to selectively deprotect the S-Mint protecting groups, removingthe solvent N₂ pressure. These bis(thiol), bis(Acm)-peptide-resinintermediates were washed with CH₂Cl₂, DMF and NMP (each 5 times×2 min,2 mL), swollen further in NMP (2 mL) for 5 min and then treated withEt₃N (2 eq.) in NMP at 25° C. for 4 h. The peptide-resin was then washedwith DMF and CH₂Cl₂ (each 5 times×2 min, 2 mL). Following resin-boundformations of the first loop, the peptide-resin was again washed withDMF (5 times×2 min, 2 mL) and swollen in DMF (2 mL) for 5 min, filteredand treated with Tl(tfa)3 (1.5 eq.) in DMF-anisole (4 mL) to cyclize thesecond disulfide loops. After gentle agitation at 25° C. for 4 h, thethallium reagents were removed with DMF (8 times×2 min, 2 mL) and thepeptide-resins were washed further with CH₂Cl₂ (5 times×2 min, 2 mL).Final cleavages of the bicyclic peptide was achieved with Reagent D(TFA:H₂O:TIS:Phenol, 87.5:5:2.5:5) (4 mL) at 25° C. for 90 min, toprovide the desired product.

The resultant conjugated peptide (SEQ ID NO:39) is shown below.

The Albumin-binding peptide-compstatin was tested forcomplement-inhibiting activity using the in vitro assay described inExample 4. As shown in FIG. 8, the conjugate was active in inhibitingcomplement activation, however, seven-fold more conjugate was requiredto achieve the same amount of inhibition as the unconjugated analog,Ac-V4(1-methyl-W)/H9A (SEQ ID NO:23).

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The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A compound that inhibits complement activation,comprising a peptide having a sequence: (SEQ ID NO: 26)Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg- Cys-Xaa5;

wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptidecomprising Gly-Ile; Xaa2 is Trp or an analog of Trp, wherein the analogof Trp has increased hydrophobic character as compared with Trp, withthe proviso that, if Xaa3 is Trp, Xaa2 is an analog of Trp comprising5-fluoro- or 6-fluoro-l-tryptophan, or a lower alkoxy or lower alkylsubstituent at the 1 or 5 position of tryptophan, or a lower alkanoylsubstituent at the 1 position of tryptophan; Xaa3 is Trp or an analog ofTrp comprising a chemical modification to its indole ring wherein thechemical modification increases the hydrogen bond potential of theindole ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile,Val, Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptidecomprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by —NH₂; andthe two Cys residues are joined by a disulfide bond.
 2. The compound ofclaim 1, wherein: (a) Xaa2 participates in a nonpolar interaction withC3; (b) Xaa3 participates in a hydrogen bond with C3; or (c) Xaa2participates in a nonpolar interaction with C3, and Xaa3 participates ina hydrogen bond with C3.
 3. The compound of claim 1, wherein the analogof Trp of Xaa2 is 5-methoxytryptophan or 5-methyltryptophan.
 4. Thecompound of claim 1, wherein the analog of Trp of Xaa2 is1-methyltryptophan or 1-formyltryptophan.
 5. The compound of claim 1,wherein the analog of Trp of Xaa3 comprises a halogenated tryptophan. 6.The compound of claim 5, wherein the halogenated tryptophan is5-fluoro-l-tryptophan, or 6-fluoro-l-tryptophan.
 7. The compound ofclaim 1, wherein Xaa4 is Ala.
 8. The compound of claim 1, wherein Xaa2comprises a lower alkanoyl or lower alkyl substituent at the 1 positionof tryptophan, Xaa3 optionally comprises a halogenated tryptophan andXaa4 is Ala.
 9. The compound of claim 8, wherein Xaa2 is1-methyltryptophan or 1-formyltryptophan and Xaa3 optionally comprises5-fluoro-l-tryptophan.
 10. The compound of claim 1, wherein the compoundis PEGylated.
 11. The compound of claim 1, further comprising anadditional peptide component that extends the in vivo retention of thecompound.
 12. The compound of claim 11, wherein the additional peptidecomponent is an albumin binding peptide.
 13. A method of making acompound that inhibits complement activation, wherein the compoundcomprises a peptide having a sequence: (SEQ ID NO: 26)Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg- Cys-Xaa5;

wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptidecomprising Gly-Ile; Xaa2 is Trp or an analog of Trp, wherein the analogof Trp has increased hydrophobic character as compared with Trp, withthe proviso that, if Xaa3 is Trp, Xaa2 is an analog of Trp comprising5-fluoro- or 6-fluoro-l-tryptophan, or a lower alkoxy or lower alkylsubstituent at the 1 or 5 position of tryptophan, or a lower alkanoylsubstituent at the 1 position of tryptophan; Xaa3 is Trp or an analog ofTrp comprising a chemical modification to its indole ring wherein thechemical modification increases the hydrogen bond potential of theindole ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile,Val, Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptidecomprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by —NH₂;wherein the method comprises synthesizing the peptide by condensation ofthe amino acid residues or analogs thereof, or expressing apolynucleotide encoding the peptide.
 14. The method of claim 13, furthercomprising cyclizing the peptide through formation of a disulfide bondbetween the two Cys residues.
 15. The method of claim 13, furthercomprising post-synthesis modification of the compound selected from oneor more of (a) acetylation of Xaa1, (b) replacement of the terminal —OHof Xaa4 with —NH₂, (3) PEGylation of the compound and (4) synthesizingthe compound with an additional peptide component that extends the invivo retention of the compound.
 16. A method of inhibiting complementactivation in or on a medium in which complement activation isoccurring, comprising contacting the medium with a complement inhibitor,wherein the complement inhibitor comprises a peptide having a sequence:(SEQ ID NO: 26) Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5;

wherein: Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptidecomprising Gly-Ile; Xaa2 is Trp or an analog of Trp, wherein the analogof Trp has increased hydrophobic character as compared with Trp, withthe proviso that, if Xaa3 is Trp, Xaa2 is an analog of Trp comprising5-fluoro- or 6-fluoro-l-tryptophan, or a lower alkoxy or lower alkylsubstituent at the 1 or 5 position of tryptophan, or a lower alkanoylsubstituent at the 1 position of tryptophan; Xaa3 is Trp or an analog ofTrp comprising a chemical modification to its indole ring wherein thechemical modification increases the hydrogen bond potential of theindole ring; Xaa4 is His, Ala, Phe or Trp; Xaa5 is L-Thr, D-Thr, Ile,Val, Gly, a dipeptide comprising Thr-Asn or Thr-Ala, or a tripeptidecomprising Thr-Ala-Asn, wherein a carboxy terminal —OH of any of theL-Thr, D-Thr, Ile, Val, Gly or Asn optionally is replaced by —NH₂; andthe two Cys residues are joined by a disulfide bond.
 17. The method ofclaim 16, wherein complement activation is inhibited in one or more of:(a) blood or serum; (b) artificial organs or implants; and (c) inphysiological fluids during extracorporeal shunting of the fluids. 18.The method of claim 16, wherein the complement activation is inhibitedas part of a treatment of a disease or condition in which complementactivation contributes to cell damage or tissue injury.
 19. The methodof claim 16, adapted to design a peptide analog or peptidomimetic or toscreen a small molecule library to identify other compounds that inhibitcomplement activation or compete with the complement inhibitor forbinding to C3 or a C3 fragment.